MGLL Human

What is the basic structure of human MGLL and how does it compare to bacterial homologs?

Human MGLL (also known as MAGL) is a serine hydrolase that catalyzes the hydrolysis of monoacylglycerol into free fatty acid and glycerol. Structurally, it contains a characteristic catalytic triad (Ser122-Asp239-His269) and an α/β-hydrolase fold. Research using X-ray crystallography has revealed unexpected conservation of the cap architecture between bacterial and human enzymes, suggesting evolutionarily preserved function . The enzyme belongs to the serine hydrolase family, with structural features optimized for monoacylglycerol binding and hydrolysis. To investigate MGLL structure, researchers typically employ X-ray crystallography, site-directed mutagenesis, and molecular modeling to elucidate structure-function relationships.

How does MGLL regulate endocannabinoid signaling in the central nervous system?

MGLL serves as the principal metabolic enzyme controlling 2-arachidonoylglycerol (2-AG) levels in the brain . As a critical regulatory point for both endocannabinoid and eicosanoid signaling pathways, MGLL catalyzes the hydrolysis of the ester bond in 2-AG, thereby terminating 2-AG-mediated signaling at CB1 and CB2 receptors while producing arachidonic acid and glycerol . This process affects numerous physiological processes including appetite, inflammation, pain sensation, and memory . Methodologically, researchers investigate this regulation using selective MGLL inhibitors (such as JZL184, KML29, or ABX-1431) to elevate 2-AG levels in vitro or in vivo, combined with lipidomic analysis to quantify endocannabinoid changes and electrophysiological methods to assess effects on neuronal signaling.

What are the analytical methods for distinguishing between MGLL activity and other serine hydrolases in human tissue samples?

Distinguishing MGLL activity from other serine hydrolases requires specialized analytical approaches:

• Activity-based protein profiling (ABPP): Uses fluorophosphonate (FP)-based probes specifically designed to target serine hydrolases. Gel-based ABPP employs fluorescent reporter groups, while mass spectrometry-based approaches use biotin as a reporter for protein identification . This technique allows assessment of enzyme activity in native states.

• Substrate-specific assays: Utilizing 2-AG or synthetic monoacylglycerol substrates with different chain lengths to exploit MGLL's substrate preference.

• Selective inhibitor profiling: Comparing enzyme activity in the presence of MGLL-selective inhibitors (JZL184, KML29, ABX-1431) versus broad-spectrum serine hydrolase inhibitors.

• Genetic approaches: Using MGLL-knockout or knockdown models as negative controls to confirm specificity of activity measurements.

• Immunocapture-activity assays: Combining immunoprecipitation with activity assays to isolate and measure MGLL-specific activity.

These methods have been crucial in developing selective MGLL inhibitors and characterizing MGLL function in human tissues .

How can activity-based protein profiling (ABPP) be optimized for MGLL research?

ABPP has become essential for MGLL research and inhibitor development. To optimize ABPP for MGLL studies:

• Probe selection: Fluorophosphonate (FP)-based probes targeting serine hydrolases are typically used, but must be carefully selected based on reactivity profiles .

• Sample preparation: Different tissue homogenization protocols may affect MGLL activity; gentle detergent solubilization preserves membrane-associated MGLL activity.

• Multiplexed analysis: Combining different ABPP probes with varied reporter groups enables simultaneous assessment of multiple enzyme classes.

• Competitive ABPP: Pre-incubating samples with inhibitors of varying concentrations establishes dose-dependent target engagement profiles.

• Quantitative mass spectrometry integration: For absolute quantification of MGLL activity across samples.

• Validation across species: Testing with multiple human proteomes and rodent brain homogenates ensures translational relevance .

This methodology was pivotal in developing selective MGLL inhibitors like ABX-1431, which has progressed to clinical studies for neurological disorders , demonstrating ABPP's value in translational research.

What strategies exist for measuring MGLL-mediated 2-AG metabolism in complex human biological matrices?

Measuring MGLL-mediated 2-AG metabolism in complex human biological matrices requires sophisticated analytical approaches:

• LC-MS/MS-based lipidomics: The gold standard for quantifying 2-AG and its metabolites in biological samples. This approach requires careful sample preparation to prevent ex vivo 2-AG degradation, including enzyme inhibitors and rapid extraction protocols.

• Ex vivo tissue activity assays: Human tissue samples can be incubated with exogenous 2-AG, with and without MGLL inhibitors, to measure enzyme activity through quantification of arachidonic acid production.

• Stable isotope tracing: Using deuterated or 13C-labeled 2-AG to track metabolism in human samples, distinguishing MGLL-mediated hydrolysis from other metabolic pathways.

• ABPP combined with mass spectrometry: Allows assessment of MGLL activity in its native state within complex proteomes .

• Microdialysis coupled to mass spectrometry: For dynamic measurements in living tissue, though primarily applicable in research settings.

These methods have demonstrated that MGLL effectively blocks 2-AG hydrolysis in human brain tissue and elevates 2-AG content in human blood without affecting stimulated prostanoid production , providing crucial human translational data.

How do experimental conditions affect measurements of MGLL kinetics, and what controls are essential?

Experimental conditions significantly impact MGLL kinetic measurements, requiring careful controls:

• pH and temperature effects: MGLL activity shows pH and temperature dependence; standardization is crucial for reproducibility. Optimal conditions (typically pH 7.2-7.4 and 37°C for human MGLL) should be established and maintained.

• Detergent and buffer composition: As a membrane-associated enzyme, MGLL activity is sensitive to detergent types and concentrations. Low concentrations of mild detergents (0.1% Triton X-100) often preserve activity while solubilizing the enzyme.

• Substrate presentation: 2-AG is highly lipophilic; its presentation affects enzyme accessibility. Micellar, liposomal, or solubilized substrate preparations yield different kinetic parameters.

• Essential controls:

  • Heat-inactivated samples to establish background

  • Selective MGLL inhibitors (JZL184, ABD-1970) to confirm specificity

  • Recombinant MGLL as positive control

  • Assessment of competing hydrolases (ABHD6, ABHD12)

  • Time-course measurements to ensure linearity

  • Substrate concentration range spanning Km

• Protein concentration optimization: Ensuring linearity of activity with respect to enzyme concentration.

These considerations are particularly important when comparing MGLL activity across different tissue sources or pathological states, or when evaluating inhibitor efficacy.

What are the critical considerations when designing selective MGLL inhibitors for human studies?

Designing selective MGLL inhibitors for human studies requires balancing several critical factors:

• Selectivity across the serine hydrolase family: MGLL belongs to a large enzyme family with similar catalytic mechanisms. Selectivity is crucial to avoid off-target effects and can be evaluated using ABPP with multiple human proteomes .

• Blood-brain barrier permeability: For CNS applications, inhibitors must penetrate the blood-brain barrier. ABX-1431 exemplifies an orally administered MGLL inhibitor with appropriate CNS pharmacokinetic properties .

• Mechanistic considerations: Covalent, irreversible inhibitors like ABX-1431 have shown efficacy , but present distinct considerations from reversible inhibitors regarding long-term dosing and safety.

• Avoiding cannabimimetic effects: Optimal inhibitors should provide therapeutic benefits without undesirable psychoactive effects associated with excessive CB1 receptor activation .

• Species differences: Human and rodent MGLL exhibit differences that may affect inhibitor binding and efficacy, necessitating human tissue validation.

Studies with ABD-1970 have demonstrated that MGLL inhibitors can provide antinociceptive and antipruritic effects without overt cannabimimetic effects , representing an important design goal.

How do rodent and human MGLL differ in their response to inhibitors, and what implications does this have for translational research?

Differences between rodent and human MGLL have significant implications for translational research:

• Structural differences: Despite high homology, subtle differences in active sites and binding pockets affect inhibitor affinity and selectivity.

• Tissue-specific arachidonic acid metabolism: MGLL contributes to arachidonic acid metabolism in a subset of rodent tissues , but these patterns may differ in human tissues, affecting downstream consequences of MGLL inhibition, particularly prostanoid production.

• Endocannabinoid system differences: CB1/CB2 receptor density, distribution, and baseline endocannabinoid levels differ between species, affecting functional outcomes of MGLL inhibition.

Methodologically, ABD-1970 has been used to define the relationship between MGLL target engagement, brain 2-AG concentrations, and efficacy in both human and rodent systems . Results show that MGLL inhibitors effectively block 2-AG hydrolysis in human brain tissue and elevate 2-AG content in human blood without affecting stimulated prostanoid production , suggesting certain aspects of pharmacology are conserved while others may be species-specific.

For translational research, these findings underscore the importance of validating MGLL inhibitors in human tissues and careful interpretation of rodent data when predicting human outcomes.

What biomarkers are most valuable for assessing MGLL inhibition in human clinical trials?

For clinical trials evaluating MGLL inhibitors, several biomarkers provide valuable insights into target engagement and pharmacological effects:

• 2-AG levels in blood or cerebrospinal fluid (CSF): Elevation of 2-AG is a direct marker of effective MGLL inhibition . Mass spectrometry-based quantification of 2-AG and related endocannabinoids provides a robust measure of pharmacodynamic effect.

• Ex vivo MGLL activity: Blood samples collected during clinical trials can be analyzed using ABPP to assess the degree of MGLL inhibition.

• Inflammatory biomarkers: Since MGLL affects production of arachidonic acid-derived inflammatory mediators, inflammatory markers provide insight into downstream effects.

• Disease-specific clinical outcome measures: For neurological disorders like Tourette syndrome, where ABX-1431 has shown potential , validated tic severity scales and quality-of-life measures are important clinical biomarkers.

• Functional neuroimaging: For CNS indications, neuroimaging techniques may provide biomarkers of neuronal activity related to altered endocannabinoid signaling.

Clinical studies with ABX-1431 have utilized multiple biomarkers to demonstrate it was well-tolerated and safe in phase 1 studies, with data indicating potential to treat symptoms in patients with Tourette syndrome .

How does MGLL function in the human immune system compared to its role in the central nervous system?

MGLL exhibits distinct yet interrelated functions in the human immune and central nervous systems:

In the central nervous system:

• Principal enzyme for degrading 2-AG, an important retrograde messenger in synaptic signaling

• Critical regulation point for endocannabinoid and eicosanoid signaling pathways

• Implicated in regulating processes like appetite, inflammation, pain sensation, and memory

• Inhibition provides antinociceptive and antipruritic effects

In the immune system:

• MGLL is induced in human monocyte-derived dendritic cells upon ligation of multiple pattern recognition receptors (PRRs)

• Regulates the abundance of secreted 2-AG, highlighting potential paracrine roles on bystander cells

• Surprisingly, inhibition of MGLL activity did not affect maturation or the production of pro-inflammatory cytokines in dendritic cells

Methodologically, these functions have been studied using selective inhibitors like JZL184 in combination with protein profiling and lipidomic analysis. Research shows that while MGLL primarily serves to terminate endocannabinoid signaling in the CNS, in immune cells it may facilitate cell-cell communication through regulation of secreted endocannabinoids .

What experimental approaches best characterize MGLL's role in neuroinflammatory processes?

Characterizing MGLL's role in neuroinflammatory processes requires multifaceted experimental approaches:

• Cell-specific genetic manipulation: Conditional knockout or knockdown of MGLL in specific cell types (microglia, astrocytes, neurons) to dissect cell-specific contributions to neuroinflammation.

• Pharmacological studies with selective inhibitors: Utilizing inhibitors like ABX-1431 , JZL184, or KML29 with readouts of neuroinflammatory markers.

• Ex vivo brain slice models: Allow manipulation of MGLL in preserved neural circuits while monitoring inflammatory responses.

• Primary human cell cultures: Studies with human microglia, astrocytes, and neurons to evaluate species-specific aspects of MGLL function.

• Multiplex cytokine/chemokine profiling: To comprehensively assess inflammatory mediator production following MGLL modulation.

• In vivo neuroimaging: Using PET tracers targeting neuroinflammatory markers combined with MGLL inhibition.

• Single-cell omics approaches: To characterize cell-type-specific responses to MGLL inhibition in neuroinflammatory contexts.

• Cerebrospinal fluid biomarker analysis: For translational studies linking MGLL inhibition to changes in neuroinflammatory markers.

These approaches have revealed that MGLL inhibition can modulate neuroinflammatory processes in multiple neurological disorders, contributing to the therapeutic potential of MGLL inhibitors for conditions like Tourette syndrome, neuromyelitis optica, and multiple sclerosis .

How does MGLL contribute to lipid metabolism in human cancer cells, and what are the implications for cancer research?

MGLL has emerged as an important regulator of lipid metabolism in human cancer cells, with significant implications for oncology research:

• Role in cancer progression: MGLL has been implicated in the progression of human melanoma, breast, ovarian, and colorectal cancer . The enzyme may facilitate metabolic adaptation supporting cancer cell growth and survival.

• Lipid metabolism remodeling: In cancer cells, MGLL can contribute to mobilization of lipid stores, providing free fatty acids for energy production, membrane synthesis, and signaling lipid generation.

• Tumor microenvironment modulation: Through regulation of endocannabinoids and eicosanoids, MGLL may influence the tumor microenvironment, affecting inflammation, angiogenesis, and immune infiltration.

• Therapeutic implications: MGLL inhibition represents a potential strategy to interfere with aberrant lipid metabolism supporting tumor growth. Studies suggest targeting MGLL could sensitize cancer cells to other therapies or directly inhibit tumor progression.

Methodologically, research in this area employs a combination of:

• Gene expression analysis to assess MGLL levels in human tumor samples

• Functional studies with genetic or pharmacological MGLL inhibition

• Lipidomics to characterize changes in lipid metabolism

• Xenograft models to evaluate impact on tumor growth in vivo

Evidence indicates MGLL may serve as a regulatory node in the altered lipid metabolism characterizing many cancers, providing new opportunities for therapeutic intervention.

How do post-translational modifications affect human MGLL activity and how can these be studied systematically?

Post-translational modifications (PTMs) of human MGLL represent a complex regulatory level affecting its activity, localization, and stability. To systematically study these PTMs:

• PTM identification: High-resolution mass spectrometry, particularly targeted proteomics approaches, can identify sites of phosphorylation, ubiquitination, acetylation, glycosylation, and other modifications. Immunoprecipitation followed by mass spectrometry (IP-MS) can enrich MGLL for detailed analysis.

• Functional impact: Site-directed mutagenesis experiments where putative PTM sites are mutated to non-modifiable or modification-mimetic residues can reveal functional impact on enzymatic activity, stability, and interactions. These mutants can be assessed using enzyme assays, ABPP, and cellular studies.

• Regulatory dynamics: Kinetic studies to determine how various cellular conditions and signals affect PTM patterning, using cell cultures exposed to different stimuli followed by temporal PTM analysis.

• Interactions with PTM machinery: Interactome studies to identify the kinases, phosphatases, ubiquitin ligases, and other modifying enzymes that regulate MGLL.

• Physiological relevance: In vivo validation studies using animal models with MGLL-specific knock-in mutations to assess the impact of PTMs on physiological functions.

Activity-based protein profiling (ABPP) is particularly valuable for this work as it allows assessment of enzymatic activity in the presence of endogenous post-translational modifications , providing a more physiologically relevant context than studies with purified recombinant proteins.

What are the molecular determinants of substrate specificity in human MGLL compared to other serine hydrolases?

The molecular determinants of substrate specificity in human MGLL involve complex structural features distinguishing it from other serine hydrolases:

• Active site architecture: The catalytic triad Ser122-Asp239-His269 resides in a binding pocket accessible through a hydrophobic channel that accommodates the substrate's fatty acid chain. Crystallographic studies reveal details about how this architecture facilitates substrate orientation for catalysis .

• Cap domain: Comparison between bacterial and human MGLL reveals unexpected conservation in cap architecture , which plays a crucial role in substrate specificity by controlling access to the active site.

• Acyl and glycerol binding sites: MGLL has distinct binding sites for the acyl and glycerol components of its substrates, enabling specificity toward monoacylglycerols rather than other lipids.

• Differences from other serine hydrolases:

  • While many serine hydrolases have broad specificity, MGLL shows preference for monoacylglycerols

  • MGLL's active site is optimized for substrates with a single fatty acid chain, unlike lipases that can accommodate multiple chains

  • The orientation of catalytic residues and shape of the binding pocket differ in ways that favor monoacylglycerols over other esters

To study these determinants, researchers employ:

• Crystallography and molecular modeling to visualize protein-substrate interactions

• Site-directed mutagenesis to test the importance of specific residues

• Kinetic analysis with diverse substrates to characterize specificity

• Comparative studies with other serine hydrolases to identify unique features

Understanding these determinants is crucial for rational design of selective inhibitors targeting MGLL without affecting other serine hydrolases.

How do the kinetics of MGLL-mediated 2-AG hydrolysis change under different cellular stress conditions, and what methodologies best capture these dynamics?

The kinetics of MGLL-mediated 2-AG hydrolysis can vary significantly under different cellular stress conditions, reflecting the adaptive nature of the endocannabinoid system. Capturing these dynamics requires sophisticated methodologies:

• Factors influencing kinetics:

  • Local substrate concentrations (2-AG levels)

  • Cell activation state and signaling

  • Changes in membrane lipid environment

  • Post-translational modifications of MGLL

  • Availability of cofactors and regulators

• Methodologies for capturing real-time dynamics:

  • FRET/BRET-based biosensors: Allow real-time monitoring of MGLL activity or 2-AG levels in living cells.

  • Microdialysis coupled to LC-MS: For dynamically measuring 2-AG levels in in vivo models.

  • Real-time kinetic enzyme assays: Using fluorogenic substrates to measure MGLL activity under different conditions.

  • Time-resolved lipidomics: Analysis of lipid profile snapshots at multiple timepoints following cellular stimulation.

  • Dynamic activity-based protein profiling (ABPP): To assess changes in MGLL activity as a function of time and condition.

• Critical methodological considerations:

  • Maintaining membrane integrity and native lipid environment

  • Minimizing artifactual 2-AG degradation during sampling and analysis

  • Controlling variables like temperature, pH, and ionic composition

  • Relating kinetic changes to functional cellular responses

Studies have shown that MGLL activity is induced following pattern recognition receptor ligation in human dendritic cells , illustrating how enzyme kinetics can change in response to cellular signals. Understanding these dynamics is crucial for predicting how MGLL inhibitors will function in different disease states and provides insight into MGLL's physiological role in various conditions.